Optically trapping confocal Raman microscopy of individual lipid vesicles: Kinetics of phospholipase A(2)-catalyzed hydrolysis of phospholipids in the membrane bilayer
ABSTRACT Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2 position of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in optically trapped liposomes is monitored in situ using confocal Raman microscopy. Individual optically trapped liposomes (0.6 microm in diameter) are exposed to PLA2 isolated from cobra (Naja naja naja) venom at varying enzyme concentrations. The relative Raman scattering intensities of C-C stretching vibrations from the trans and gauche conformers of the acyl chains are correlated directly with the extent of hydrolysis, allowing the progress of the reaction to be monitored in situ on a single vesicle. In dilute vesicle dispersions, the technique allows the much higher local concentration of lipid molecules in a single vesicle to be detected free of interferences from the surrounding solution. Observing the local composition of an optically trapped vesicle also allows one to determine whether the products of enzyme-catalyzed hydrolysis remain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lag prior to the rapid hydrolysis. The lag time varied inversely with the enzyme concentration, which is consistent with the products of enzyme-catalyzed lipid hydrolysis reaching a critical concentration that allows the enzyme to react at a much faster rate. The turnover rate of membrane-bound enzyme determined by Raman microscopy during the rapid, burst-phase kinetics was 1200 s(-1). Based on previous measurements of the equilibrium for PLA2 binding to lipid membranes, the average number of enzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quite small, only two PLA2 molecules at the lowest enzyme concentration studied.
Article: Vesicle PhotonicsAnnual Review of Materials Research 07/2013; 43(1):283-305. DOI:10.1146/annurev-matsci-071312-121724 · 15.63 Impact Factor
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ABSTRACT: Interfaces and surfaces play very important roles in many chemical, physical and biological processes. In order to reveal the molecular structures on the surface or interface at a molecular level, one needs effective interfacial techniques. As a second order non-linear optical technique, sum frequency generation (SFG) vibrational spectroscopy, which has extremely high surface selectively and sensitivity, intrinsically meets the requirement. Due to the coherent property of SFG, the interference effects are always involved in observed SFG signals. The quantitative understanding and analysis of the interference effects on SFG spectra are critical for resolving molecular structure from SFG measurement. However, a general model dealing with the interference effect on the SFG spectra is still not widely available yet. In this thesis, a general model has been developed to quantitatively treat the interference effects in the SFG spectra for thin-layer systems, especially those from the different vibrational modes, different interfaces and those from the interference in the thin-film with change of film thickness. The validity of the modeling simulations has been verified by the experimental observations. The model calculation can also help us to optimize the experimental conditions for the thin-film systems. The modeling calculation has also been successfully applied to study the adsorption of solvent molecules on the surface of the LiCoO2 thin film, which is an important cathode material for lithium ion batteries, to further improve its efficiency and life-time. Finally, the modeling calculation was employed to investigate the hydrolysis process of supported lipid bilayer systems catalyzed by a phospholipase A2 enzyme. A novel hydrolysis reaction mechanism has been proposed based on the analysis on the SFG observation.03/2010, Degree: PhD, Supervisor: Prof. Shen Ye
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ABSTRACT: Sum mothers do ′ave ′em: Sum‐frequency generation spectroscopy was used to investigate the hydrolysis mechanism of a dipalmitoylphosphatidylcholine bilayer by phospholipase A2 (PLA2). This study describes the structural changes and the hydrolysis mechanism in each leaflet (red and black) of a supported lipid bilayer at a molecular level.Angewandte Chemie International Edition 03/2010; 49(13):2319-23. DOI:10.1002/anie.200904950 · 11.34 Impact Factor